Production of tumor angiogenesis factor by cell culture

ABSTRACT

Process for the production of human TAF in vitro comprising growing the human Burkitt lymphoma cell line Raji in agitated, liquid suspension of nutrient culture medium at about 35°-38° C. for a sufficient time to elaborate TAF and recovering the resulting TAF from the cells or cell product.

BACKGROUND OF THE INVENTION

This invention relates to the in vitro production of human tumorangiogenesis factor from the human Burkitt lymphoma cell line Raji.

It has been known for some time that unless a solid tumor is providedwith blood vessels by its host it remains small and dormant. Thesubstance that is released by tumors and provides vascularization hasbeen named tumor angiogenesis factor (TAF) by Dr. Judah Folkman of theHarvard Medical School. J. Exptl. Med. 133, 275-88 (1971); Cancer Res.34, 2109-13 (1974).

Ever since it was recognized that tumor induced neovascularizationrepresents the breaching of an important barrier in the control of tumorgrowth, considerable effort has been spent in searching for ways toinhibit neovascularization. It was early suggested by Dr. Folkman thatblockade of this factor (inhibition of angiogenesis) might arrest solidtumors at a tiny diameter of a few millimeters (avascular phase). NewEngl. J. Med. 285, 1182 (1971); J. Exptl. Med. 133, 275-88 (1971). Onesuggested approach was the raising of antibodies against TAF extractsfor the production of an anti-serum. Folkman, Ann. Surg. 175 409-16(1972); Phillips et al., Int. J. Cancer 17, 549-58 (1976). Anotherapproach to inhibiting neovascularization which has received muchattention recently consists in treatment of tumor cells with extracts ofcartilage. Eisenstein et al, Am. J. Pathol. 73, 765-74 (1973); Sorgenteet al., Lab. Invest. 32, 217-22 (1975); Eisenstein et al., Amer. J.Pathol. 81, 337-48 (1975); "Medical News", JAMA 232 (1), 14-15 (1975);Brem and Folkman, J. Exptl. Med. 141, 427-39 (1975); and Kuettner etal., U.S. Pat. No. 4,042,457. These efforts are limited, of course, tothe amount of TAF which is available and suitable for test purposes.

TAF finds further use in the development of tests such as an angiogenicassay or a diagnostic screening test for neoplasia. Klagsbrun et al.,Cancer Res. 36, 110-14 (1976); and Brem et al., Science 195, 880-81(1977); Cancer 41, 239-44 (1978).

TAF also has been proposed as useful for wound healing, Rettusa et al,FASEB, Abstract No. 4309, 61st Ann. Meet., Apr. 1-8, 1977, Chicago, Ill.

Various human tumor cells have been reported heretofore as capable ofelaborating TAF when measured by certain assays. Thus, extracts fromhuman neoroblastoma, Wilms' tumor and human hepatoblastoma were found tocontain TAF which was able to cause the formation of new blood vesselsin the subcutaneous fascia of rats. Folkman et al., J. Exptl. Med. 133,275-88 (1971).

So also, WI-38 embryonic lung, SV W126 (SV 40 virus transformed W126),glioblastoma, meningioma and HeLa cells (the latter only in suspensionculture and not in monolayers) grown in T-75 tissue culture flasks weredescribed as giving a positive TAF response as measured by bioassay onthe chick chorioallantoic membrane (CAM). Folkman and Klagsbrun, Chapter31 of "Fundamental Aspects of Neoplasia," at page 402, (Gottlieb et al,eds.), Springer-Verlag, New York, 1975.

Subsequently, extracts from hypernephroma, haemangioma and human kidneywere similarly described as eliciting a TAF response which resulted inthe growth of new capillaries in the subcutaneous fascia of rats.Phillips et al., Int. J. Cancer 17, 549-58 (1976).

In still other experiments, Hubler and Wolf demonstrated TAF responsesfrom human cutaneous melanoma implanted in transparenthamster-cheek-pouch membranes. Cancer 38, 187-92 (1976).

All of the foregoing tumor cells are derived from fresh tumors orprimary cultures except the WI-38, SV W126 and HeLa cells. Fresh tumorsand primary cultures are not, however, generally suitable sources of TAFexcept for limited research purposes or small scale production. In orderto provide a commercially significant source of TAF in terms of readyavailability and adequate supply, production from a suitable establishedcell line is deemed necessary. As a practical matter, the cell lineshould have not only specific TAF activity, but it should also have goodcell growth characteristics in terms of rapid growth, good suspensiongrowth, adaptability to large-scale culture and economical nutrientrequirements.

The terms "cell line" and "established cell line" are used herein inconformance with the definitions published by Federoff in the TissueCulture Association Manual, Vol. 1, No. 1, pp. 53-57 (1975).

DESCRIPTION OF THE INVENTION

Applicants have investigated numerous human tumor cell lines for theproduction of TAF, but most of them have been eliminated as unsuitablecandidates in view of their poor growth characteristics asabove-defined.

One cell line that has now been found to have good growthcharacteristics is suspension culture and is able to elaborate thedesired TAF in suitable quantities is the human Burkitt lymphoma cellline Raji. The established cell line Raji was derived from a Burkittlymphoma by R. J. V. Pulvertaft in 1963. Cultures of this cell line areavailable from the American Type Culture Collection (Rockville, Md.)under the code number ATCC CCL 86. The establishment andcharacterization of this Burkitt lymphoma cell line is further describedby Pulvertaft, Lancet 1, 238-40 (1964), and Epstein et al., J. Natl.Cancer Inst. 37, 547-59 (1966). Other publications on cell line Rajiinclude the following:

Argyios et al., J. Exptl. Med. 139, 696-711 (1974); and

Minowada et al., Cancer Res. 34, 1898-1903 (1974).

Although Raji cells have been studied in detail and have been known tobe able to synthesize interferon, this cell line has not heretofore beenknown as a suitable source of TAF.

Initially, the medium used by applicants for maintenance and growth ofthis cell line was Dulbecco's modification of Eagle's minimum essentialmedium (MEM) containing 4 mg/ml of glucose. The culture media wassupplemented with 15% by volume fetal bovine serum without addition ofany antibiotics, although lower concentrations of fetal bovine serum,for example, about 5-10%, also can be used. These cells of lymphoidorigin were grown at 37° C. in agitated, liquid suspension culture in4-liter vessels analogous to the procedure described in U.S. Pat. No.4,059,485. The cells were then harvested after times ranging from six toeight days growth since inoculation, and TAF was extracted and assayedaccording to the general procedures described in U.S. Pat. No.4,059,486.

Specifically, the cells after growth were washed successively threetimes in lactated Ringer's solution, resuspended in phosphate bufferedsaline (pH 7-7.4) and stirred for 4 hours at 4° C. The supernatant afterremoval of the cells by centrifugation was retained as the cell extract.The cells pellet from the extract was resuspended in distilled water andstirred at 37° C. for 20 minutes. The supernatant after removal of thecell debris by centrifugation was retained as the cell lysate. In onerun of 4 liters (after 8 days growth) the cell extract was assayed tocontain 49.7 mg. of total protein (Lowry protein assay) while the celllysate was assayed to contain 610 mg. of total protein. In another runof 4 liters (after 6 days growth) the cell extract contained 71.2 mg. oftotal protein and the cell lysate contained 526 mg. of protein. The cellextracts from these two runs gave strongly positive tests in thebioassay for TAF in the chorioallantoic membrane (CAM) of chick embryos,in accordance with test procedure described by Folkman, Cancer Res. 34,2109-13; 36, 110-14 (1976).

In subsequent runs, the Raji cells were grown succesfully in a 40-literflask employing a continuous cell culture system as described incopending application Ser. No. 850,987, filed Nov. 14, 1977 and assignedto a common assignee. After 6 days of growth at 37° C. in Dulbecco's MEMcontaining 4 mg/ml. of glucose and supplemented with 15% fetal bovineserum, the cells were harvested and the TAF was extracted as above. Thecell extract from 36 liters of suspension was assayed to contain 2.3grams of total protein while the cell lysate was assayed to contain 17.9grams of total protein. The cell extracts were then subjected to furtherpurification by CM-Sephadex® (Pharmacia) ion exchange chromatography andlyophilized. The purified material also gave a positive test in thebioassay for TAF. The use of CM-Sephadex chromatography for obtaining aTAF active fraction from tumor cells is described by Tuan et. al.,Biochemistry 12 (17), 3159-65 (1973).

It will be appreciated that other nutrient culture media for culture ofthe Raji cells can be used in place of Dulbecco's MEM, for example, anyof the well-known tissue culture media described by H. J. Morton, InVitro 6, 89-108 (1970). These conventional culture media contain knownessential amino acids, mineral salts, vitamins and carbohydrates. Theyare also frequently fortified with mammalian sera such as fetal bovineserum. Suitable growth of the cells can be carried out at about 35°-38°C. but cell proliferation is best at 37° C. Growth under these cellculture conditions for about 4-8 days generally is sufficient to producethe desired TAF.

Other suitable equipment and procedures for growing cells in agitated,liquid suspension which can be adapted for culture of the cell line Rajiwill be readily apparent to the person skilled in the art by referenceto well-known texts on cell culture such as, for example, Paul, "Celland Tissue Culture," the Williams and Wilkins Company, Baltimore, 4thed. 1970, at pages 277-291; Kruse and Patterson, "Tissue Culture Methodsand Applications," Academic Press, New York, 1973, at pages 333-377.

It should also be understood that various means can be used to separateand purify TAF-containing fractions from the cells and cell cultureproduct other than the illustrative specific recovery proceduresdescribed above. These various recovery means include the knowntechniques for the separation and purification of proteinaceoussubstances in general such as, for example, dialysis, salt and solventprecipitation, adsorption with gels, cellulose ion exchangechromatography, Sephadex gel filtration, electrophoresis, andlyophilization. Thus, TAF can be obtained by extraction from the cellsfollowed by subjecting the extract to dialysis against NaCl (0.15 M),Sephadex G-100 chromatography, dialysis against water and lyophilizationanalagous to the methods described by Folkman et al., J. Exptl. Med.133, 275-88 (1971) and Phillips et at., Int. J. Cancer 17, 549-58(1976). According to Phillips et al., id, when the dialysate issubjected to chromatography on a column of Sephadex G-100 in 0.15 MNaCl, the fractions between 35,000 and 300,000 molecular weight containthe greatest TAF activity. According to Folkman, Cancer Res. 34, 2109-13(1974), TAF is a proteinaceous substance having a molecular weight ofapproximately 100,000. It will be appreciated, however, that theaforesaid molecular weights are estimates based on partially purifiedTAF-containing products and the inventors are not bound by suchextimates or by any particular purification procedure or TAF purity.

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention. All such further examples are includedwithin the scope of the appended claims.

What is claimed is:
 1. Process for the production of human TAF in vitro comprising growing the human Burkitt lymphoma cell line Raji in agitated, liquid suspension of nutrient culture medium at about 35°-38° C. for a sufficient time to elaborate TAF and isolating the resulting TAF from the cells or cell product.
 2. The process of claim 1 in which the nutrient culture medium is Dulbecco's modification of Eagle's minimum essential medium.
 3. The process of claim 1 in which the nutrient culture medium is fortified with mammalian serum.
 4. The process of claim 1 in which the nutrient culture medium is fortified with from about 5% to about 15% fetal bovine serum.
 5. The process of claim 1 in which the TAF is isolated by extraction from the cells.
 6. The process of claim 1 in which the nutrient culture medium is Dulbecco's modification of Eagle's minimum essential medium and is fortified with fetal bovine serum and in which the TAF is isolated by extraction from the cells. 